US 11,692,196 B2
Methods for creating synthetic chromosomes expressing biosynthetic pathways and uses thereof
Edward Perkins, Savannah, GA (US); and Amy Greene, Savannah, GA (US)
Assigned to CarryGenes Bioengineering, LLC, Savannah, GA (US)
Appl. No. 16/92,837
Filed by SynPloid Biotek, LLC, Savannah, GA (US)
PCT Filed Apr. 11, 2017, PCT No. PCT/US2017/027069
§ 371(c)(1), (2) Date Oct. 11, 2018,
PCT Pub. No. WO2017/180665, PCT Pub. Date Oct. 19, 2017.
Claims priority of provisional application 62/321,716, filed on Apr. 12, 2016.
Prior Publication US 2021/0403930 A1, Dec. 30, 2021
Int. Cl. C12N 15/81 (2006.01); C12N 15/85 (2006.01)
CPC C12N 15/81 (2013.01) [C12N 15/85 (2013.01)] 12 Claims
 
1. A method for constructing a mammalian synthetic chromosome to express genes encoding enzymes involved in the synthesis of metabolites in a biosynthetic pathway in a host cell comprising:
(a) transfecting a satellite DNA-based mammalian synthetic chromosome production cell line with synthetic chromosome production components comprising nucleic acids engineered to incorporate multiple site-specific integration sites into a satellite DNA-based synthetic platform chromosome as it is produced in the satellite DNA-based mammalian synthetic chromosome production cell line;
(b) identifying and isolating an engineered mammalian cell carrying the satellite DNA-based mammalian synthetic platform chromosome comprising multiple site-specific integration sites produced in (a);
(c) transfecting the isolated engineered mammalian cell of (b) carrying the synthetic platform chromosome with a delivery vector comprising at least one site-specific recombination site and multiple genes encoding multiple gene products in a first biosynthetic pathway, wherein multiple site-specific recombination sites and integration sites are selected from attachment site pairs comprising target DNA sequences attB/attP and attL/attR, or mutated versions of attP/attB and attL/attR;
(d) activating site-specific recombination between the synthetic platform chromosome and the delivery vector using an integrase, wherein the multiple genes encoding multiple gene products in the first biosynthetic pathway are loaded onto the synthetic platform chromosome in the engineered cell to produce a satellite DNA-based mammalian synthetic chromosome expressing multiple gene products that act in concert in the first biosynthetic pathway; and
(e) isolating a first host cell comprising the satellite DNA-based mammalian synthetic chromosome, wherein the first host cell expresses the multiple gene products that act in concert to produce metabolites in the first biosynthetic pathway.